People are increasingly aware of the lack of ability of 2D cell culture to predict the complex behavior of a biological system of many different interacting cell types and to reproduce the anatomy or physiology of a tissue for informative or useful study. 3D cell culture models are a more accurate representation of the natural environment experienced by the cells in the living organism, which allows for intercellular interactions with more realistic biochemical and physiological responses. In 3D cell cultures, cells behave and respond more like they would in vivo to internal and external stimuli, such as changes in temperature, pH, nutrient absorption, transport, and differentiation. Therefore, scientists are shifting their focus from 2D to 3D cell cultures in the fields of drug screening, tissue engineering, preclinical study, cell therapy, and basic cell biological study.
To mimic in vivo cell growing conditions, the reticulated structure of 3D scaffold should be serialized, have a high water content, and have a number of other desirable characteristics such as accurate 3D spatial support, suitable mechanical strength, and facile transportation of oxygen, nutrients, waste, and soluble factors. Mild and cytocompatible conditions for sol-gel transformation are preferred, to ensure that cells survive comfortably during both cell encapsulation and isolation. Moreover, the injectable property of biomaterial used for 3D cell culture is critical for downstream applications such as cancer therapy (xerography study for drug discovery), tissue regeneration, and 3D bio-printing.
The current materials for 3D cell cultures on the market can be classified as hydrogels, polymer matrices, hanging drop plates, low adhesion plates, micro-patterned surfaces, and magnetic levitations. Hydrogel scaffolds have been demonstrated as the most promising approach to date in facilitating 3D cell culture. However, most existing biomaterials (including hydrogel scaffolds) for 3D cell cultures are limited to physiological conditions (e.g. poor scaffold structure, unwanted growth factors, and undesirable pH or temperature of pre-gel solution), complex operating steps for cell encapsulation, difficulties for cell isolation from culture scaffold, and product reproducibility. In addition, injectable properties, such as shear-thinning and rapid recovery of physical strength, in currently marketed hydrogel materials is very rare. This drawback not only affects the data generated from these 3D cell culture technologies but also limits the applications of this technology for downstream analysis and clinical applications. Examples of related art are described below:
U.S. Patent Application No. 2008/0220526 pertains to coatings for cell culture surfaces. More particularly, this invention relates to coatings for cell culture surfaces which are derived from or contain gums including naturally occurring gums, plant gums, galactomannan gums or derivatives thereof. The invention also relates to articles of manufacture (e.g., cell culture vessels and labware) having such coatings, methods of applying these coatings to cell culture surfaces, and methods of using coated cell culture vessel.
U.S. Pat. No. 9,579,417 pertains to cell-adhesive gellan gum spongy-like hydrogels that are able to entrap/encapsulate adherent cells, which spread within the material, maintaining their phenotype and remaining viable and proliferative. The methodology used to obtain these materials involves hydrogel preparation, freezing, freeze-drying and re-hydration with a saline solution with cells and with/without bioactive molecules. No pre and/or post functionalization of the spongy-like hydrogels with cell adhesive features, as used for other hydrogels, is used. The cell adhesive character of these materials, not observed in hydrogels, is in part explained by their physical properties, between sponges and hydrogels, dissimilar from the precursor hydrogels. The physical properties that are mainly different are the morphology, microstructure, water content, and mechanical performance. Gellan gum spongy-like hydrogels physical properties and biological performance can be tuned by manipulating the parameters involved in spongy-like hydrogel formation. Bioactive molecules can also be entrapped with or without cells to modify the biological performance of the spongy-like hydrogels. These materials can be applied in the context of bioengineering, tissue engineering, regenerative medicine and biomedical applications.
Chinese Patent Application No. 106474560 pertains to the technical field of biological material, discloses for 3D biological printing of the hydrogel material and its preparation method and application. Hydrogel material of this invention comprises the following mass percentage component: and/or its derivatives to form the 0.5-10%, PEG and/or its derivatives 0.1-20%, cross-linking initiator 0-1%, biological active component 0-15%, the rest of the solvent. The invention is based on the form the hydrogel material and PEG double-network hydrogel, physiological environment forming an interpenetrating double-network structure, has better structure and size stability, has fast gel under physiological conditions, cell with good biocompatibility, immune rejection small, cell encapsulation rate high, the mechanical strength of the controllable, biodegradable and the like. And applied to the 3D in biological printing, overcomes the slow curing speed, curing conditions are harsh, mechanical property is limited, cells poor compatibility, has obvious advantages and good industrialization prospects.
PCT Patent Application No. WO2014025312 pertains to a method of manufacturing hydrogel microparticles comprising one or more species of living cells attached thereon and/or encapsulated therein is provided. The method includes dissolving a hydrogel-forming agent in an aqueous medium to form a solution; suspending one or more species of living cells in the solution to form a cell suspension; dispersing the cell suspension into an organic oil to form a microemulsion; and subjecting the microemulsion to conditions that allow the hydrogel-forming agent to form hydrogel microparticles comprising one or more species of living cells attached thereon and/or encapsulated therein. Composition comprising a mixture of a degradable hydrogel and at least one hydrogel microparticle having one or more species of living cells, and method of manufacturing a scaffold for tissue engineering are also provided.
PCT Patent Application No. 2014017513 pertains to a method for culturing a cell and/or a tissue, said method being characterized by culturing the cell and/or the tissue in a floated state using a culture medium composition, wherein amorphous structures are formed in a liquid culture medium, are dispersed in the solution uniformly, and substantially hold the cell and/or the tissue without substantially increasing the viscosity of the solution, so that the culture medium composition has an effect of preventing the sedimentation of the structures; and others.
None of the art described above addresses all of the issues addressed by the embodiments of the present invention. There clearly exists an unmet need for finding compositions and methods for preparing gellan gum hydrogel systems that are suitable for cell culture and various biomedical applications, including but not limited to, 2D coating culture, 3D cell culture, and injection.